© 2016. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
CLINICAL PUZZLE
The role of enterocyte defects in the pathogenesis of congenital
diarrheal disorders
ABSTRACT
Congenital diarrheal disorders are rare, often fatal, diseases that are
difficult to diagnose (often requiring biopsies) and that manifest in the
first few weeks of life as chronic diarrhea and the malabsorption of
nutrients. The etiology of congenital diarrheal disorders is diverse, but
several are associated with defects in the predominant intestinal
epithelial cell type, enterocytes. These particular congenital diarrheal
disorders (CDDENT) include microvillus inclusion disease and
congenital tufting enteropathy, and can feature in other diseases,
such as hemophagocytic lymphohistiocytosis type 5 and
trichohepatoenteric syndrome. Treatment options for most of these
disorders are limited and an improved understanding of their
molecular bases could help to drive the development of better
therapies. Recently, mutations in genes that are involved in normal
intestinal epithelial physiology have been associated with different
CDDENT. Here, we review recent progress in understanding the
cellular mechanisms of CDDENT. We highlight the potential of animal
models and patient-specific stem-cell-based organoid cultures, as
well as patient registries, to integrate basic and clinical research, with
the aim of clarifying the pathogenesis of CDDENT and expediting the
discovery of novel therapeutic strategies.
KEY WORDS: Brush border, Cell polarity, Congenital diarrheal
disorder, Enterocyte, Intracellular trafficking, Microvillus inclusion
diseases
Introduction
Congenital diarrheal disorders (CDDs) are a group of rare inherited
intestinal disorders that are characterized by persistent life-threatening
intractable diarrhea and nutrient malabsorption, which emerge during
the first weeks of life. The etiology of CDDs is diverse, including
defects in enteroendocrine cells, dysregulation of the intestinal
immune response, or defects in the predominant cell type of the
intestinal epithelium, the enterocyte (Canani et al., 2015).
CDDs associated with enterocyte defects (abbreviated as
CDDENT) include disorders that can be treated with nutrition
therapy. Other CDDENT require life-long total parenteral nutrition
(TPN; see Box 1 for a glossary of clinical terms used in this article)
to receive adequate nutrition, and are a leading indication for
pediatric intestinal transplantations (Halac et al., 2011). Most
CDDENT are difficult to diagnose, and clinical management is
restricted to the treatment of symptoms; there is currently no cure. If
left untreated, CDDENT are invariably fatal.
Owing to the consanguinity of parents of affected children, genetic
defects associated with CDDENT have recently been identified. The
clinical consequences of some mutations – those affecting specific
transporter proteins or certain enzymes – are relatively
straightforward, such as in individuals with congenital lactase
deficiency or sucrase-isomaltase deficiency caused by loss-offunction mutations in lactase (Behrendt et al., 2009; Kuokkanen
et al., 2006) and sucrase-isomaltase (Ritz et al., 2003), respectively.
Other mutations, however, are in genes that have less well-understood
functions in intestinal epithelial physiology, such as in individuals
with microvillus inclusion disease (MVID), congenital tufting
enteropathy (CTE), familial hemophagocytic lymphohistiocytosis
type 5 (FHL5) and trichohepatoenteric syndrome (THES) (Fabre
et al., 2012; Hartley et al., 2010; Heinz-Erian et al., 2009;
Müller et al., 2008; Sivagnanam et al., 2008; Szperl et al., 2011;
Wiegerinck et al., 2014; zur Stadt et al., 2009). Table 1 summarizes
CDDENT-associated genes, the proteins they encode and their
function. Understanding the mechanisms by which these mutations
lead to disease should pinpoint targets for improved diagnosis and
therapeutic intervention.
The identification of genetic mutations in individuals with
CDDENT has confirmed the autosomal recessive inheritance pattern
of these diseases; thus, genetic counselling and prenatal diagnosis
are important tools for heterozygote carriers. Because the
histological hallmarks that characterize some CDDENT can be
very subtle and easily missed, the identification of genetic defects
contributes to a better and faster differential diagnosis, which is
currently offered by several medical centers worldwide.
Here, we discuss the different CDDENT, recent discoveries
concerning their underlying molecular and genetic mechanisms,
and the model systems used in researching these disorders. Further,
basic research is urgently needed to improve the diagnosis and
management of these devastating diseases, and for developing new
therapeutic strategies to combat them.
Enterocytes: a brief overview
1
Department of Cell Biology, University Medical Center Groningen, University of
2
Groningen, 9713 AV Groningen, The Netherlands. Department of Pediatrics and
Adolescent Medicine, University Medical Center Ulm, 89075 Ulm, Germany.
3
Department of Pediatrics, Erasmus Medical Center Rotterdam, Erasmus University
4
Rotterdam, 3000 CB Rotterdam, The Netherlands. Department of Pediatrics,
Leiden University Medical Center, Leiden University, 2300 RC Leiden,
The Netherlands.
*Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Enterocytes are the absorptive cells in the lining of the intestinal
mucosa. Enterocytes originate from the intestinal stem cells that
reside in the intestinal crypts (Sato et al., 2009), and differentiate
and migrate within 3-4 days from the crypt to the villus tip, where
they are extruded into the gut lumen. Enterocytes are arranged as a
monolayer of polarized epithelial cells (Fig. 1) (Massey-Harroche,
2000). Their plasma membrane consists of a basal and a lateral
domain, facing the underlying tissue and neighboring cells,
respectively, and an apical domain, facing the gut lumen. Densely
packed microvilli, supported by an actin filament meshwork,
1
Disease Models & Mechanisms
Arend W. Overeem1, Carsten Posovszky2, Edmond H. M. M. Rings3,4, Ben N. G. Giepmans1 and
Sven C. D. van IJzendoorn1, *
CLINICAL PUZZLE
Box 1. Clinical glossary
Aminoaciduria: a disorder of protein metabolism in which excessive
amounts of amino acids are excreted in the urine.
Atresia: the congenital absence, or the pathological closure, of an
opening, passage, or cavity.
Bowel rest: the intentional restriction of oral nutrition.
Chronic diarrhea: the passage of three or more loose or liquid stools per
day for more than 2-4 weeks.
Hepatomegaly: enlargement of the liver.
Hypercalciuria: the presence of abnormally high levels of calcium in the
urine; usually the result of excessive bone loss in hyperparathyroidism or
osteoporosis.
Hypobetalipoproteinemia: a hereditary disorder characterized by low
levels of beta-lipoproteins, lipids and cholesterol.
Hypocholesterolemia: the presence of abnormally small amounts of
cholesterol in the circulating blood.
Intractable diarrhea: treatment-resistant, non-infectious diarrhea with
high mortality; need for total parenteral nutrition. Intractable diarrhea of
infancy is a heterogeneous syndrome with different etiology.
Intrahepatic cholestasis: obstruction within the liver that causes bile
salts, bile pigments and lipids to accumulate in the bloodstream.
Metabolic acidosis: a clinical disturbance characterized by an increase
in plasma acidity.
Punctate keratitis: a condition characterized by a breakdown or
damage of the epithelium of the cornea in a pinpoint pattern.
Siderosis: a form of pneumoconiosis due to the inhalation of iron
particles.
Total parenteral nutrition (TPN): intravenous feeding that provides
patients with all the fluid and the essential nutrients they need when
feeding by mouth is inhibited.
Trichothiodystrophy: an autosomal recessive inherited disorder
characterized by brittle hair and intellectual impairment.
Woolly hair: unusually curled hair.
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
Defects of brush-border-associated enzymes and
transporter proteins
The majority of CDDENT are caused by autosomal recessive
mutations in genes that encode brush-border-associated enzymes
and transporter proteins (Canani et al., 2015) (Table 1). Depending on
the type of mutation, these proteins are either not expressed, not
correctly transported to the brush border membrane, or display
defects in their activity, resulting in defective digestion, absorption
and/or transport of nutrients, metabolites and/or electrolytes at the
enterocyte brush border. Subsequent changes in the concentration of
osmotically active compounds in the gut lumen cause diarrhea.
Prototypical examples of these CDDENT are glucose-galactose
malabsorption (caused by mutations in the Na+/glucose
cotransporter gene, SGLT1) (Martín et al., 1996), congenital lactase
deficiency (mutations in the lactase gene, LCT) (Kuokkanen et al.,
2006), sucrase-isomaltase (SI) deficiency (caused by mutations in the
SI gene) (Ritz et al., 2003), congenital chloride diarrhea (caused by
mutations in the solute carrier family 26 member 3 gene, SLC26A3)
(Wedenoja et al., 2011); several other CDDENT can also be included
in this category (Canani and Terrin, 2011) (Table 1).
Individuals with familial diarrhea syndrome have activating
mutations in GLUCY2C, which encodes the guanylate cyclase 2C
protein. Mutated guanylate cyclase 2C enhances cellular cGMP
levels (Fiskerstrand et al., 2012). cGMP stimulates cystic fibrosis
transmembrane conductance regulator (CFTR) activity in the brush
border of enterocytes by stimulating its proper translocation,
resulting in enhanced secretion of chloride and water (GolinBisello et al., 2005). CDDENT associated with functional defects of
brush-border-associated enzymes and transporter proteins are
typically not associated with abnormal enterocyte organization, as
examined by histology.
Defects in intracellular protein transport
Diverse molecular mechanisms of CDDENT underlying
clinical presentation and diagnosis
Based on recent molecular and cell biological studies, enterocyte
defects that underlie CDDENT can be divided into defects of
(i) brush-border-associated enzymes and transporter proteins;
(ii) intracellular protein transport; (iii) intracellular lipid transport
and metabolism; and (iv) intestinal barrier function (Table 1).
2
In other CDDENT, apical brush-border-associated enzymes and
transporter proteins are collectively mislocalized in the enterocytes,
indicative of general defects in intracellular protein transport.
Examples of CDDENT characterized by this class of defect are
described below.
Microvillus inclusion disease
Individuals with MVID suffer from persistent diarrhea, nutrient
malabsorption and failure to thrive (Cutz et al., 1989). In most cases
(95%), symptoms develop within days after birth, but a late-onset
variant, which manifests 2-3 months postnatally, has also been
described (Cutz et al., 1989). Variable extra-intestinal symptoms
include intrahepatic cholestasis and renal Fanconi syndrome (van
der Velde et al., 2013) (see MVID case study in Box 2). Some
individuals with MVID present less-severe digestive symptoms for
reasons that are not clear (Perry et al., 2014).
MVID, which is diagnosed by intestinal biopsy, features villus
atrophy, microvillus atrophy, and the redistribution of CD10 and
periodic acid Schiff (PAS)-stained material from the brush border
to intracellular sites (Phillips et al., 2000) in the enterocytes.
Staining of the epithelial cell-cell adhesion protein EpCAM,
aberrant in CTE, is normal (Martin et al., 2014). A definitive
diagnosis is recommended prior to potential intestinal
transplantation, and this includes analysis by electron
microscopy (EM) for microvillus inclusions in the cytoplasm of
enterocytes. The frequency of such inclusions can be very low
and repeated rounds of EM analyses can be required, although
semi-automated EM might help to increase the efficiency of
screening (de Boer et al., 2015). Immuno-based detection of
Disease Models & Mechanisms
protrude from the apical surface, resulting in a brush border
appearance. Microvilli increase the absorptive surface area of the
cells and release small vesicles that contribute to epithelialmicrobial interactions (Crawley et al., 2014; Shifrin et al., 2012).
The plasma membrane domains are equipped with distinct enzymes
and transporter proteins that control the metabolism, absorption and/
or secretion of nutrients, metabolites and electrolytes between the
gut lumen, cell interior and body tissue. The polarized distribution
of these proteins at the different plasma membrane domains is
secured by their intracellular sorting and trafficking via the Golgi
apparatus and endosomes (van der Wouden et al., 2003; Weisz and
Rodriguez-Boulan, 2009). Tight junctions between the apical
domain and the lateral surface domain provide tight intercellular
adhesion, which limits protein diffusion between the apical and
lateral plasma membrane domains, and controls the paracellular
transport of electrolytes and water (Giepmans and van Ijzendoorn,
2009; Marchiando et al., 2010). Adherens junctions in the lateral
domain mediate cell-cell adhesion strength (Giepmans and van
Ijzendoorn, 2009). Enterocyte polarity and cell-cell adhesion
junctions together provide the selectively permeable barrier
function of the intestinal epithelial monolayer.
CLINICAL PUZZLE
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
Table 1. Overview of CDDENT, their associated genes and currently available model systems
Disease name
Gene
Protein
Cell models
Animal models
Patientspecific
models
Glucose-galactose
malabsorption
SGLT1
Na+/glucose
cotransporter
Glucose and
galactose
transport
–
SGLT1 KO mouse
–
Congenital lactase deficiency
LCT
Lactase
Lactose
digestion
–
–
–
Sucrase-isomaltase (SI)
deficiency
SI
Sucraseisomaltase
Carbohydrate
digestion
–
–
–
Congenital chloride diarrhea
SLC26A3
Solute carrier
family 26
member 3
Chloride and
bicarbonate
exchange
–
SLC26A3 KO mouse
(Wang et al., 2004)
–
Familial diarrhea syndrome
GUCY2C
Guanylate cyclase
2C
Enterotoxin
receptor
–
GUCY2C KO mouse
–
Acrodermatitis enteropathica
SLC39A4
Solute carrier
family 39
member 42
Zn2+ transport
–
Conditional and global
SLC39A4 KO mouse
–
Fanconi-Bickel syndrome
SLC2A2
Solute carrier
family 2 member
2
Glucose
transport
–
SLC2A2 KO mouse
–
Lysinuric protein intolerance
SLC7A7
Solute carrier
family 7 member
7
Amino acid
transport
–
SLC7A7 KO mouse
–
Maltase-glucoamylase
deficiency
MGAM
Maltaseglucoamylase
Carbohydrate
digestion
–
MGAM KO mouse
–
Primary bile acid diarrhea
SLC10A2
Solute carrier
family 10
member 2
Bile acid
transport
–
SLC10A2 KO mouse
–
MVID
MYO5B
Myosin Vb
Molecular motor,
plasma
membrane
recycling
Caco-2 cells (RNAi)
(Dhekne et al.,
2014; Knowles
et al., 2014)
Myo5B KO mouse;
Rab8A KO mouse;
conditional Rab11a
KO mouse;
conditional Cdc42
KO mouse (CartónGarcía et al., 2015;
Melendez et al.,
2013; Sakamori
et al., 2012; Sato
et al., 2007;
Sobajima et al.,
2014).
Explant culture
(Rhoads
et al., 1991)
Atypical MVID
STX3
Syntaxin-3
Apical plasma
membrane
fusion
Caco-2 cells (mutant
Stx3
overexpression)
(Wiegerinck et al.,
2014)
–
Intestinal
organoid
(Wiegerinck
et al., 2014)
FHL5
STXBP2
Munc18-2
Apical plasma
membrane
fusion
–
–
–
THES
TTC37
TRP37
–
–
–
SKIV2L
Helicase SKI2W
mRNA
processing
and decay
mRNA
processing
and decay
–
–
–
SAR1B
Sar1b
–
Sar1b-knockdown
zebrafish (Levic
et al., 2015)
–
CMRD
Protein
function
Chylomicron
transport
vesicle fusion
Continued
3
Disease Models & Mechanisms
Model systems
CLINICAL PUZZLE
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
Table 1. Continued
Model systems
Protein
function
Patientspecific
models
Disease name
Gene
Protein
Cell models
Animal models
Familial hypobetalipoproteinemia
APOB
ApoB
Chylomicron
formation
–
Modified ApoB allele in
mouse
–
Abetaliproteinemia
MTTP
Microsomal
triglyceride
transfer protein
Acyl CoA:
diacylglycerol
acyltransferase 1
Chylomicron
formation
–
Conditional and global
MTTP KO mouse
–
Triglyceride
synthesis
–
DGAT1 KO mouse
–
Cell-cell
adhesion,
differentiation
and
proliferation
Serine protease
inhibitor
T84 cells (RNAi)
(Kozan et al.,
2015)
Global EPCAM KO
mouse (Guerra et al.,
2012; Kozan et al.,
2015)
–
–
–
–
DGAT1
CTE
EPCAM
EpCAM
SPINT2
Kunitz-type serine
protease
inhibitor
CMRD, chylomicron retention disease; CTE, congenital tufting enteropathy; FHL5, familial hemophagocytic lymphohistiocytosis type 5; KO, knockout; MVID,
microvillus inclusion disease; THES, trichohepatoenteric syndrome.
villin, which marks microvillus inclusions, has been proposed to
be a useful adjunct in MVID diagnosis (Shillingford et al., 2015).
Notably, microvillus inclusions are also present in rectal biopsies,
facilitating diagnosis if a duodenal biopsy is not feasible. Some
individuals with clinical symptoms typical of MVID show no
microvillus inclusions but do show the other enterocyte
Lumen
TJ
β-cat
EpCAM
Villus
Lys
NHE-3
LE
AEE
NHE-2
BEE
CRE
CD10
Nucleus
Rab11a MyoVb
Rab8
vSNARE
Brush
border
ARE
STX3
Munc18-2
Apical vesicle
Ezrin
Golgi
TJ
NIS
AQP7
Microvillus
Apical
membrane
Basal
membrane
Lamina
propria
Crypt
Fig. 1. Schematic overview of tissue and cellular characteristics of healthy intestinal epithelium and enterocytes. In healthy enterocytes, the apical
recycling endosome (ARE; green) is located sub-apically, and is important for transporting apically residing proteins (depicted in red) to the apical membrane
(brush border), via mechanisms that are not well understood that involve the small GTPases Rab11a and Rab8, and the effector protein myosin Vb (MyoVb; see
text). At the apical membrane, syntaxin-3 (STX3) and Munc18-2 (STXBP2) are involved in the fusion of the membrane-bound apical vesicle (orange). β-catenin
(β-cat) and EpCAM mediate cell-cell adhesion. Other organelles and common trafficking routes are shown in light gray (not discussed here). AEE, apical early
endosome; AQP7, aquaporin-7; BEE, basolateral early endosome; CRE, common recycling endosome; LE, late endosome; Lys, lysosome; NHE, sodium/
hydrogen exchanger; NIS, Na/I symporter; TJ, tight junction.
4
Disease Models & Mechanisms
H/K ATPase
abnormalities, suggesting that MVID is a heterogeneous disease
(Mierau et al., 2001).
MVID and variants of MVID are associated with MYO5B,
STXBP2 and STX3 mutations (Table 1) (Müller et al., 2008;
Ruemmele et al., 2010; Stepensky et al., 2013; Szperl et al., 2011;
Wiegerinck et al., 2014). Deletion of the Myo5B gene in mice
Box 2. Case study: MVID presenting with renal Fanconi
syndrome
A boy born to unrelated parents, born at term by spontaneous vaginal
delivery after an uncomplicated pregnancy, was hospitalized 2 months
after birth because of dehydration, metabolic acidosis, feeding
intolerance and intractable diarrhea. The diarrhea persisted during
fasting and showed elevated stool sodium content consistent with
secretory diarrhea. He was given total parenteral nutrition (TPN) via a
central venous line. Exhaustive etiological investigations ruled out
infectious or allergic etiologies. Duodenum biopsies were taken and
processed for light microscopy and electron microscopy (EM)
examination. A moderate degree of villus atrophy, and partial
intracellular periodic acid Schiff (PAS) and CD10 staining were
observed. EM revealed moderate brush border atrophy but it took
three rounds of examination before microvillus inclusions were found,
and the diagnosis of MVID was accordingly made. The patient was
discharged on home TPN. When hospitalized for the evaluation of
growth failure, excessive urinary losses of phosphate were observed
without rapid catch-up of weight gain. Examination showed severely
reduced tubular phosphate resorption, hypercalciuria, generalized
aminoaciduria and severe rickets, which are characteristics of renal
Fanconi syndrome. No disturbances in glomerular function were
observed. Phosphorus in the parenteral nutrition was increased
stepwise and treatment with oral phosphate was added. The parenteral
and oral supplementation of phosphate resulted in a gradual increase in
serum phosphate levels, a decrease of alkaline phosphatase, a
normalization of the bone density and resolution of his rickets. Also,
catch-up growth was obtained. Laboratory results indicated that the
persistence of renal Fanconi syndrome gradually resolved after the
patient received a multi-organ transplant (small intestine, large intestine,
pancreas and liver) at the age of 5 years, and enteral feeding was fully
restored. Examination of kidney biopsies from this patient revealed no
intracellular PAS staining in the proximal tubular epithelial cells and, at
the ultrastructural level, proximal tubular epithelial cells showed a normal
apical brush border. This patient illustrates the clinical complications and
underscores the need for reliable genotype-phenotype correlations to
understand the extra-intestinal clinical symptoms.
causes the development of early-onset MVID (Cartón-García
et al., 2015). MYO5B encodes the actin-based motor protein
myosin Vb, which consists of an N-terminal actin-binding motor
domain and a C-terminal tail domain that includes the cargobinding domain. Based on crystal structures of the myosin Vb
protein, mutations in MYO5B have been functionally categorized
(van der Velde et al., 2013). The myosin Vb cargo-binding
domain binds selectively to small Rab GTPases, including
RAB11A and RAB8A. Myosin Vb, RAB11A and RAB8A
associate with apical recycling endosomes (AREs) in polarized
epithelial cells, where they control the activity of the small
GTPase CDC42 (Bryant et al., 2010), and both myosin Vb and
RAB11A are mislocalized in MVID enterocytes (Fig. 2) (Dhekne
et al., 2014; Szperl et al., 2011). Accordingly, in addition to
Myo5B knockout (KO) mice (Cartón-García et al., 2015), mice in
which the intestinal Rab8a, Rab11a or Cdc42 genes have been
individually deleted also develop the cellular hallmarks of MVID
(Melendez et al., 2013; Sakamori et al., 2012; Sato et al., 2007;
Sobajima et al., 2014). However, diarrhea is not observed in
Rab11a or Cdc42 KO mice, and Rab8a KO mice survive for
approximately 5 weeks after birth, thus more closely resembling
the phenotype of late-onset MVID. Mutagenesis of residues in
myosin Vb that mediate this protein’s interaction with either
RAB11A or RAB8A, and the subsequent introduction of these
mutant forms into myosin Vb-silenced human Caco-2 cells
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
(Caucasian colon adenocarcinoma), revealed that the uncoupling
of myosin Vb from both RAB11A and RAB8A forms the basis of
MVID pathogenesis (Knowles et al., 2014).
Rab11a- and Rab8a-positive AREs play a pivotal role in
epithelial polarity development (Bryant et al., 2010; Golachowska
et al., 2010; Overeem et al., 2015; Wakabayashi et al., 2005).
Rab11a-positive AREs localize in close proximity to the apical
brush border surface in enterocytes and harbor signaling molecules,
including: phosphoinositide-dependent protein kinase-1 (PDK1)
(Dhekne et al., 2014; Kravtsov et al., 2014); the PDK1 target,
atypical protein kinase C-iota; and the ezrin-phosphorylating
kinase, Mst4 (Dhekne et al., 2014). Myosin Vb is required for the
polarized, subapical localization of Rab11a-positive AREs (Szperl
et al., 2011), which, in turn, is required for efficient Mst4-mediated
phosphorylation of ezrin and for ezrin-controlled microvilli
development (Dhekne et al., 2014). Myosin-Vb-controlled AREs
might thus function as a subapical signaling platform that regulates
the absorptive surface area of enterocytes (Dhekne et al., 2014).
Interestingly, ezrin depletion in the mouse intestine leads to a
disorganized subapical actin filament web and causes microvillus
atrophy (Saotome et al., 2004), similar to that seen in individuals
with MVID. The presence of ezrin at the intestinal brush border
correlates with the expression and function of the Na+/H+ hydrogen
exchanger (NHE)-3, which regulates sodium absorption, and loss of
Nhe-3 in mice leads to diarrhea (Ledoussal et al., 2001). MVID
enterocytes show reduced NHE-3 expression (Ameen and Salas,
2000), and MVID jejunal explants revealed a net secretory state of
the jejunum (Rhoads et al., 1991).
The ectopic expression of the myosin Vb tail domain, which acts
as a dominant-negative mutant by competing with endogenous
myosin Vb for the Rab proteins, can disrupt the delivery of proteins
from Rab11a-positive AREs to the apical plasma membrane
(Golachowska et al., 2010). The mechanism by which myosin Vb
controls apical-surface-directed transport of proteins from AREs is
not fully understood. Interestingly, individuals with mutations in
either STX3 (Wiegerinck et al., 2014), which encodes the
transmembrane protein syntaxin-3, or STXBP2 (Stepensky et al.,
2013), which encodes Munc18-2, develop the clinical symptoms
and cellular characteristics of MVID. Mutations in STX3 or STXBP2
give rise to disorders termed atypical MVID and FHL5, respectively
(Fig. 2). In enterocytes, syntaxin-3 resides at the apical cell-surface
domain, where it, in concert with SNAP23 and Munc18-2, mediates
the fusion of transport vesicles with the apical plasma membrane
(Riento et al., 2000). MVID-associated STX3 mutations cause the
depletion of syntaxin-3 or the expression of a syntaxin-3 protein that
lacks the transmembrane domain (Wiegerinck et al., 2014),
disrupting its function. STXBP2 mutations abolish the interaction
of Munc18-2 with syntaxin proteins (zur Stadt et al., 2009).
Interestingly, enterocytes of conditional Rab11 KO mice show
altered localization of syntaxin-3 (Knowles et al., 2015). It is
possible that myosin Vb mediates the apical trafficking of syntaxin3 via AREs, and protein delivery to the apical cell surface. However,
the effect of myosin Vb mutations on the apical membrane fusion
machinery in MVID remains to be demonstrated. It should be noted
that a homozygous mutation in STX3 was also reported in an
individual with autosomal recessive congenital cataracts and
intellectual disability phenotype, without mention of intestinal
symptoms (Chograni et al., 2015); thus, further investigation into
genotype-phenotype correlation of the different STX3 mutations is
warranted.
Taken together, the available data suggest that defects in ARE
function result in brush border microvillus atrophy and in the
5
Disease Models & Mechanisms
CLINICAL PUZZLE
CLINICAL PUZZLE
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
MVID
A
Atypical MVID
B
FHL5
Ezrin
MI
MI
CD10
CD10
CD10
NHE-3
EpCAM
TJ
β-cat
TJ
Rab11 MyoVb
Rab8
CD10
TJ
Lateral
microvillus
CD10
ARE
Ezrin
NHE-3
CD10
CD10
vSNARE
Munc18-2
Stx3
vSNARE
Stx3
Munc18-2
Lamina propria
intracellular retention of enzymes and transporters that are required
for the absorption of nutrients and ions by villus enterocytes,
leading to the clinical phenotype of malabsorption and diarrhea in
MVID (Dhekne et al., 2014; Knowles et al., 2014) (Fig. 2).
Trichohepatoenteric syndrome
Individuals with THES present with intractable diarrhea in the first
months of life accompanied by nutrient malabsorption and failure to
thrive (Hartley et al., 2010). THES is associated with facial
dysmorphism, hair abnormalities and, in some cases, skin
abnormalities and immune disorders (Goulet et al., 2008). Some
individuals with THES display trichothiodystrophy, liver disease,
hepatomegaly and siderosis (see Box 1). Affected individuals are
prone to infections, might fail to produce antibodies upon vaccination,
or present with low immunoglobulin levels. Mild intellectual
deficiency is a feature of ∼50% of all cases. THES can present as
very-early-onset inflammatory bowel disease (Kammermeier et al.,
2014). It is diagnosed on the basis of its clinical features and via
biopsies of the small intestine, which reveal villus atrophy, variable
immune cell infiltration of the thin layer of loose connective tissue that
lies beneath the epithelium (called the lamina propria), and no specific
histological abnormalities of the epithelium.
THES is associated with TTC37 or SKIV2L mutations. TTC37
encodes the tetratricopeptide repeat protein 37. SKIV2L encodes
SKI2 homolog, superkiller viralicidic activity 2-like protein, which
might be involved in antiviral activity by blocking translation of poly
(A)-deficient mRNAs. In enterocytes with TTC37 mutations, the
brush-border-associated NHE-2 and -3, aquaporin-7, the Na+/I−
symporter, and the H+/K+-ATPase show reduced expression or
mislocalization to the apical cytoplasm, with different patterns of
mislocalization relative to their normal pattern (Hartley et al., 2010).
NHE-2 and NHE-3 play an important role in salt and water
absorption from the intestinal tract, and loss of Nhe3 in the mouse
6
intestine causes mild diarrhea (Ledoussal et al., 2001). In THES
enterocytes, the brush border appears normal at the ultra-structural
level, as does the basolateral localization of Na+/K+-ATPase
(Hartley et al., 2010). Loss of TTC37 results in the defective
trafficking and/or decreased expression of apical transport proteins,
including aquaporin-7 (Fig. 3). The expression and distribution of
apical transporters have not yet been analyzed for individuals with
THES with SKIV2L mutations. The gene products of both TTC37
and SKIV2L are human homologs of components of the yeast Ski
complex, which is involved in exosome-mediated degradation of
aberrant mRNA and associates with transcriptionally active genes
(Fabre et al., 2012). TTC37, but not SKIV2L, is highly co-expressed
with two genes involved in apical trafficking (SCAMP1 and EXOC4;
http://coxpresdb.jp/cgi-bin/coex_list.cgi?gene=9652&sp=Hsa2). The
mechanism underlying THES is currently unknown, so further studies
are needed to elucidate potential relationships between TTC37/
SKIV2L, the Ski complex and the trafficking of apical transporter
proteins.
Interestingly, another tetratricopeptide repeat protein, TTC7A, is
implicated in a different disorder: multiple intestinal atresia (MIA).
Stem-cell-derived intestinal organoids from a MIA individual show
enterocyte polarity defects that are rescued by pharmacological
inhibition of the small GTPase RhoA (Bigorgne et al., 2014;
Overeem et al., 2015). Although MIA is not a CDD, these findings
further accentuate the role of tetratricopeptide-repeat proteins in
functional enterocyte polarity and associated intestinal disorders.
Defects in intracellular lipid transport and metabolism
In addition to defects in the intracellular transport of proteins,
defects in the intracellular transport of lipids, summarized in Fig. 4,
have been associated with CDDs. Our current understanding of the
molecular mechanisms underlying this class of CDDs is
summarized below.
Disease Models & Mechanisms
Fig. 2. Schematic overview of tissue and cellular defects associated with MVID and FHL5. (A) In typical microvillus inclusion disease (MVID), which is
caused by loss of MyoVb in the apical recycling endosome (ARE; green), villi are shortened, and microvilli are shortened and fewer in number (see Fig. 1 for
comparison). The normally apically localized proteins Ezrin, NHE-3 and CD10 are mislocalized in microvillus inclusions (MIs) or in unknown intracellular
compartments (gray, dotted lines). The ARE is localized near the nucleus instead of sub-apically. (B) In familial hemophagocytic lymphohistiocytosis type 5
(FHL5; right-hand panel), microvilli are shortened, whereas, in atypical MVID (left-hand panel), microvilli are both shortened and fewer in number. Loss of
syntaxin-3 (STX3), as occurs in atypical MVID, or of Munc18-2, as occurs in FHL5, inhibits the fusion of vesicles with the apical membrane, resulting in the
intracellular retention of apical proteins (demonstrated here for CD10). Additionally, the formation of MIs and of lateral microvilli occurs in atypical MVID, but not in
FHL5. β-cat, β-catenin; MyoVb, myosin Vb; NHE, sodium/hydrogen exchanger; STX3, syntaxin-3; TJ, tight junction.
CLINICAL PUZZLE
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
TJ
NHE-3
NHE-2
AQP7
H/K ATPase
TTC37
NIS
TJ
Lamina propria
Chylomicron retention disease
Individuals with chylomicron retention disease (CMRD) suffer
from chronic diarrhea, severe lipid malabsorption, failure to thrive,
and hypocholesterolemia as a result of by hypobetalipoproteinemia.
Large lipid vacuoles and chylomicron-like particles retained within
membrane-bound compartments, which could represent prechylomicron transport vesicles, are typically observed in the
cytoplasm of CMRD enterocytes. Microvilli appear normal by
EM examination (Mouzaki et al., 2014).
CMRD is caused by mutations in SAR1B (Jones et al., 2003). The
Sar1b protein is part of the Sar1-ADP-ribosylation factor family of
small GTPases and triggers the formation of coat protein complex II
(COPII)-coated transport vesicles from the endoplasmic reticulum
(Fig. 4). In CMRD, SAR1B mutations result in defective trafficking
of nascent chylomicrons in pre-chylomicron transport vesicles
between the endoplasmic reticulum and the Golgi apparatus, thereby
interfering with the successful assembly of chylomicrons and their
delivery to the lamina propria (Mansbach and Siddiqi, 2010). It
remains unclear how defective intracellular chylomicron trafficking
results in intestinal lipid malabsorption and diarrhea. Sar1 proteins
are also involved in the trafficking of CFTR (Wang et al., 2004),
which is a typical brush border protein in enterocytes. In the fruit fly
Drosophila melanogaster, Sar1b is involved in the trafficking of
Crumbs (Kumichel et al., 2015), a protein that controls apical-basal
epithelial cell polarity also in the intestine (Whiteman et al., 2014).
Whether SAR1B mutations in CMRD also affect the trafficking of
apical brush border proteins in enterocytes and thereby contribute to
impaired (lipid) absorption remains to be investigated.
Familial hypobetalipoproteinemia and abetaliproteinemia
Two other CDDENT have been associated with defects in
intestinal fat absorption and chylomicron assembly. Familial
Fig. 4. Schematic overview of the cellular processes involved in lipid
transport and metabolism in enterocytes. After uptake from the lumen, fatty
acids (FAs) and monoacylglycerol (2MG) are transported to the endoplasmic
reticulum (ER) (1). Here (see magnified view), they are converted to
triglycerides (TGs) in several metabolic steps (not shown), the last of which is
dependent on DGAT1 (2). ApoB and MTTP act in concert to incorporate
triglycerides into a chylomicron (yellow) (3). The newly formed chylomicron
buds from the ER in a prechylomicron transport vesicle (PCTV) (4), which
subsequently fuses with the Golgi, a process that is dependent on Sar1b (5).
The chylomicron is then transported in a vesicle to the basal membrane, where
it exits the cell (6). FA, fatty acid; 2MG, sn-2-monoacylglycerol; CoA, coenzyme
A; DG, diacylglycerol; MTTP, microsomal triglyceride transfer protein; PCTV,
prechylomicron transport vesicle; TG, triglyceride; TJ, tight junction.
hypobetalipoproteinemia (FHBL), the only CDDENT that is
dominantly inherited, is associated with mutations in the APOB
gene, encoding apolipoprotein B (Young et al., 1990), which,
together with triglycerides and other lipids, makes up the nascent
chylomicron (Fig. 4). Abetaliproteinemia is associated with
mutations in the MTTP gene, which encodes microsomal
triglyceride transfer protein (MTTP). MTTP catalyzes the
transfer of triglycerides to nascent ApoB particles in the
endoplasmic reticulum. Abetaliproteinemia-associated mutations
reduce MTTP activity, the synthesis of very-low-density
lipoproteins, and lipid absorption in the intestine. To date, there
have been two known cases of congenital diarrhea associated with
mutations in DGAT1, which encodes acyl CoA:diacylglycerol
acyltransferase 1, an enzyme that is involved in triglyceride
synthesis and is highly expressed in the intestine (Fig. 4) (Haas
et al., 2012). The mechanism by which DGAT1 mutations cause
diarrhea is unclear, but is likely to involve the build-up of DGAT1
lipid substrates in the enterocytes or in the gut lumen (Haas et al.,
2012). Dgat1 KO mice do not develop diarrhea, and it has been
proposed that this is due to compensatory Dgat2 expression in the
mouse intestine (Buhman et al., 2002). The observation that the
overexpression of Sar1b in human Caco-2 cells stimulated DGAT
and MTTP activity (Levy et al., 2011) underscores the fact that all
currently known CDDENT that are associated with defective lipid
absorption originate in defects in the triglyceride-rich lipoprotein
assembly pathway.
7
Disease Models & Mechanisms
Fig. 3. Schematic overview of tissue and cellular defects associated with
THES. In individuals with trichohepatoenteric syndrome (THES), villus or
microvillus defects are not observed. Through an unknown mechanism, lossof-function mutations in TTC37 result in the intracellular localization of the
normally apically localized H+/K+-ATPase, the Na/I symporter (NIS), and the
apical proteins NHE-2 and NHE-3. These mutations also result in the loss of
expression, either global or local, of certain apical proteins, such as aquaporin7 (AQP7). NHE, sodium/hydrogen exchanger; TJ, tight junction.
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
Defects in intestinal barrier function
The barrier function of the intestine is important for fluid
homeostasis and critically depends on cell-cell adhesions. Defects
in the intestinal barrier function have been associated with at least
one CDD.
Congenital tufting enteropathy
Congenital tufting enteropathy (CTE) is characterized by persistent
diarrhea that presents immediately or shortly after birth, despite
bowel rest and total parenteral nutrition (TPN) (Goulet et al., 2007).
Some affected individuals display a milder phenotype than others,
and these can sometimes be progressively weaned off TPN (Lemale
et al., 2011). A subset of individuals with CTE display a syndromic
form of the disease [congenital sodium diarrhea (CSD)] that
includes dysmorphic features, woolly hair, punctate keratitis,
atresias, reduced body size and immune deficiency (see Box 1).
Like THES, CTE can present as very-early-onset inflammatory
bowel disease (Kammermeier et al., 2014).
Histological analysis of the intestine in the context of CTE reveals
various degrees of villous atrophy, basement membrane
abnormalities, disorganization of enterocytes, and focal crowding
at the villus tips, resembling tufts (Fig. 5). There is no evidence for
abnormalities in epithelial cell polarization; the enterocyte brush
border appears normal, and the staining pattern of the brush-borderassociated metallopeptidase CD10 is normal (Martin et al., 2014),
but expression of desmogleins, a family of cadherins, is enhanced
(Goulet et al., 2007). The major diagnostic marker is the absence of
epithelial cell adhesion molecule (EpCAM) staining in CTE
enterocytes (Martin et al., 2014). Furthermore, immune cell
infiltration into the lamina propria is absent. In some cases,
however, increased numbers of inflammatory cells have been
reported in the lamina propria, indicating that their presence does
not preclude the diagnosis of CTE (Kammermeier et al., 2014).
TJ
Tuft
CD10
EpCAM
β-cat
TJ
Lamina propria
Fig. 5. Schematic overview of tissue and cellular defects associated with
CTE. In congenital tufting enteropathy (CTE), villi are shortened and are
disorganized, with focal crowding of enterocytes (tufts). Mutated EpCAM is
mislocalized intracellularly, which results, through an unknown mechanism, in
the loss of tight junction (TJ) integrity and a concomitant increase in
permeability (red arrows). TJ, tight junction; β-cat, β-catenin.
8
CTE is associated with EPCAM or SPINT2 mutations. EpCAM is
a multifunctional transmembrane glycoprotein involved in cell-cell
adhesion, proliferation and differentiation (Schnell et al., 2013b). In
individuals with CTE, EpCAM protein levels in the intestine are
decreased (Sivagnanam et al., 2008) and all CTE-associated
EPCAM mutations lead to loss of cell-surface EpCAM (Fig. 5)
(Schnell et al., 2013a), either because of impaired plasma membrane
targeting or because of truncation of the protein, both of which
result in its secretion. Both Epcam KO mice and mice in which exon
4 of Epcam is deleted develop CTE (Guerra et al., 2012; Kozan
et al., 2015). In the Epcam KO mouse intestine, E-cadherin and
β-catenin, two adherens-junction-associated proteins, are also
mislocalized, leading to disorganized transition from crypts to
villi (Guerra et al., 2012). Mice with reduced EpCAM levels and
Caco-2 cells depleted of EpCAM show decreased expression of
tight-junction proteins, increased permeability and decreased ion
transport (Kozan et al., 2015). EpCAM interacts with the tightjunction proteins claudin-7 and claudin-1 (reviewed in Schnell
et al., 2013b). Conceivably, loss of EpCAM expression and/or
function leads to the increased permeability of the intestinal barrier
by disrupting tight junctions (Fig. 5), resulting in diarrhea.
The mechanism by which mutations in SPINT2 lead to CTE
phenotypes, however, is not clear. SPINT2 encodes the
transmembrane Kunitz-type 2 serine-protease inhibitor. Spint2
KO mice are embryonically lethal owing to developmental defects
that are unrelated to the intestine (Szabo et al., 2009), and are
therefore unsuitable for studying the intestinal symptoms of CTE.
Interestingly, two of the target enzymes of Spint2 are the serine
proteases matriptase and prostasin (Szabo et al., 2009), which are
primary effector proteases of tight-junction assembly in intestinal
epithelial cells (Buzza et al., 2010). The Y163C mutation in Spint2
results in a complete loss of the ability of Spint2 to inhibit prostasin
and another intestinal protease, the transmembrane protease serine
13 (Tmprss13) (Faller et al., 2014). Further investigation is needed
to determine the role of Spint2 and other proteases in the regulation
of cell-cell junctions in the pathogenesis of CTE.
The inhibition of trypsin-family serine peptidases, such as that
encoded by SPINT2, abolishes the constitutive stimulation of apical
Na+ transport by nonvoltage-gated sodium channel-1-alpha
(Scnn1a) in polarized intestinal epithelial cells (Planès and
Caughey, 2007), which could contribute to secretory diarrhea. It
is possible that such a mechanism forms the basis of the syndromic
form of congenital sodium diarrhea that is associated with SPINT2
mutations (Faller et al., 2014; Heinz-Erian et al., 2009).
Publically available bioinformatics gene co-expression
databases show that the EPCAM and SPINT2 genes are strongly
co-expressed in humans (http://coxpresdb.jp/cgi-bin/coex_list.
cgi?gene=4072&sp=Hsa, and http://coxpresdb.jp/cgi-bin/coex_
list.cgi?gene=10653&sp=Hsa), which suggests that they either
share a transcriptional regulatory program, are functionally related,
or are members of the same pathway or protein complex.
Interestingly, ST14, the gene that encodes matriptase, is strongly
co-expressed with both EPCAM and SPINT2, further underscoring
the need to study its involvement in the pathogenesis of CTE.
Outlook and future perspectives
Establishing a molecular diagnosis for CDDENT is becoming feasible
in most cases, and can be a key contributor to clinical decision
making. At the moment, the prognosis and survival of individuals
with CDDENT depend on early TPN and successful bowel
transplantation, but survival is generally poor. A variety of extraintestinal symptoms are associated with CDDENT. Of these, renal
Disease Models & Mechanisms
CLINICAL PUZZLE
CLINICAL PUZZLE
Fanconi syndrome in MVID disappears after bowel transplantation
(Golachowska et al., 2012), whereas intrahepatic cholestasis in
MVID is aggravated after bowel transplantation (Girard et al., 2014;
Halac et al., 2011). It remains unclear whether these symptoms are
iatrogenic, i.e. complications of treatment, and/or are linked to
particular CDDENT-associated gene mutations or the genetic
background of the patient. Prospective patient registries, animal
models, and stem-cell-based organoid technology combined with
novel gene-editing tools, such as CRISPR, will address these current
shortcomings in our knowledge, as discussed below (see Box 3).
Patient registries and databases
Dedicated patient registries are crucial resources for correlating the
genotype, phenotype and clinical presentation of individuals with
CDDENT. Thus far, only a registry of patients with MVID and
associated MYO5B mutations has been established (http://www.
mvid-central.org) (van der Velde et al., 2013). Given that
individuals with CDDENT display partially overlapping
phenotypes, the expansion of such a database to include other
CDDENT patients, including a prospective set-up that allows the
course of disease to be recorded together with the influence of
therapeutic interventions, is expected to improve disease diagnosis,
prognosis and counseling.
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
intestine. In developing larvae of the fly Drosophila melanogaster,
myosin-V deficiency interferes with apical protein secretion in the
hindgut (Massarwa et al., 2009). This suggests a problem with
apical protein delivery and warrants further research to examine the
potential of myosin-V-deficient flies as a model for CDDENT. Other
CDDENT-associated genes have not yet been examined in worms or
flies.
The ability to perform high-throughput assays and intravital
imaging in vertebrate zebrafish (van Ham et al., 2014) make
these animals a promising model for studying the effect of
genetic manipulations and pharmacological treatment. Intestinal
anatomy and architecture in zebrafish closely resemble the
anatomy and architecture of the mammalian small intestine
(Yang et al., 2014) and have been used to study enteropathies
such as congenital short bowel syndrome (Van Der Werf et al.,
2012). Zebrafish could therefore make a useful addition to
current CDDENT models. Indeed, sar1b-deficient zebrafish
display phenotypes resembling CMRD (Levic et al., 2015).
The absence of the myosin-V ortholog in zebrafish results in an
abnormal epidermal tissue structure. In the study reporting this
mutant, inclusion bodies in the intestine are mentioned (Sonal
et al., 2014). epcam-deficient zebrafish have aberrant epidermal
development; however, intestinal defects have not been reported
(Slanchev et al., 2009).
Vertebrate and invertebrate model organisms for CCDENT
Box 3. Clinical and basic research opportunities
•
•
•
•
•
The use of genetics and automated microscopy in the differential
diagnosis of CDDENT in the clinic.
The development of genetically engineered animal models of CDDENT.
The creation of CDDENT patient-specific stem-cell-based organoids for
disease modeling.
The establishment and maintenance of CDDENT patient registries that
integrate basic and clinical data.
The exploration of stem-cell-based replacement strategies as a
potential cure for CDDENT.
Stem-cell-based organoids
Advances in stem cell technology provide new models for studying
CDDENT. Generating three-dimensional cultures of stem-cellderived intestinal cells that resemble to some extent the intestinal
tissue (so-called organoids) enables disease modeling that better
resembles the in vivo situation while still retaining experimental
versatility and the ability to genetically manipulate cells. Organoids
allow for patient-specific personalized disease modeling.
Promisingly, intestinal organoids generated from STX3-mutationcarrying individuals with MVID recapitulate most of the in vivo
phenotypes (Wiegerinck et al., 2014).
Intestinal organoids can be generated from adult stem cells and by
differentiating induced pluripotent stem cells (iPSCs) into intestinal
cell types (Forster et al., 2014; Sato et al., 2009; Spence et al., 2011).
Although both adult-stem-cell- and iPSC-derived intestinal tissue
structures are referred to as organoids, notable differences exist
between the two. Organoids obtained from iPSCs, but not from
adult stem cells, contain supporting mesenchymal cells. Moreover,
iPSC-derived organoids are relatively immature with fetal-like
characteristics, although transplantation of iPSC-derived immature
organoids under the kidney capsule of mice results in the
development of mature, engrafted intestinal tissue that develops
villi and crypts (Watson et al., 2014). From adult stem cells, only
genomically engineered organoids that contain tumorigenic
mutations have undergone successful engraftment under the
mouse kidney capsule, suggesting that mesenchymal cells are
required for organoid maturation outside of the intestinal niche
(Matano et al., 2015). However, adult-stem-cell-derived organoids
have been reported to engraft in the chemically injured mouse colon,
to contribute to tissue regeneration, and to be indiscernible from
host epithelium (Yui et al., 2012).
These differences are important to consider when organoids are
used to study CDDENT. The investigation of phenotypes that
manifest at a multicellular level, such as the structural villi
abnormalities in MVID and CTE, requires a model that forms
villi and crypts. The maturity of organoids is also relevant because
CDDENT phenotypes do not always manifest immediately after birth
9
Disease Models & Mechanisms
Intestinal epithelial cell lines cannot recapitulate all of the
phenotypes associated with CDDENT, such as those related to the
different states of proliferation and differentiation in enterocytes as
they migrate from the crypts to the villus tips in the intestine. This is
important for understanding the cellular defects seen in MVID and
CTE, which are more pronounced in the villus than they are in the
crypt region (Groisman et al., 1993; Phillips et al., 2000; Thoeni
et al., 2014). Cell lines also do not form villi, precluding the study of
villi defects, villus atrophy and villus tufts. Finally, studies in
intestinal cell lines do not take into account effects beyond the
intestine.
Animal models offer a useful system for determining causal
relationships between genes and CDDENT, for investigating disease
pathogenesis, and for evaluating treatment options preclinically. KO
animals are useful for studying the function of the targeted gene and
for modeling CDDENT individuals with homozygous mutations, and
gene-editing techniques such as CRISPR-Cas can be used to introduce
patient-relevant homozygous and compound heterozygous missense
mutations both in animal and cell-line models.
The potential use of model organisms other than mice for
CDDENT research has not been fully explored. Intestinal brush
border proteins are normally apically localized in invertebrate
nematode Caenorhabditis elegans worms that lack Hum2, the
ortholog of MYO5 (Winter et al., 2012). Conceivably, this reflects
the distinct physiology and cellular architecture of the worm
(e.g. late-onset forms). A practical consideration is that adult-stemcell-derived organoid culture requires invasive biopsies, whereas
the somatic cells to generate iPSCs can be non-invasively acquired.
Organoid technology uniquely allows the creation of patientspecific disease models. Despite harboring mutations in the same
protein, many individuals with CDDENT often vary in the range and
severity of their symptoms. This suggests that different mutations
could have a varying effect on protein function, and thus on disease
outcome. Other potential factors that could influence such variation
are the genetic background of a patient and any adverse effects of
treatment. Organoids from affected individuals with varying
symptoms exclude confounding environmental factors and
provide a model in which phenotypes are tissue-autonomous and
solely dependent on patient genotype. The use of gene-editing tools,
such as CRISPR, in organoid cultures could provide a valuable tool
for making definitive genotype-phenotype correlations. Finally,
organoids created from different organs of the same patient could
provide additional insights into the genetic relationship of extraintestinal symptoms associated with CCDENT.
Although diagnostic tools for CCDENT have improved over the
last few years, a cure for CDDENT is desperately needed. Organoid
transplantation and/or cell-replacement strategies can lead to the
restoration of the intestinal epithelium in mice (Yui et al., 2012).
This raises the exciting possibility of investigating whether
CRISPR-based correction of mutations in patient stem cells and
transplantation of genetically corrected organoids could represent a
regenerative medicine approach to cure CDDENT.
Acknowledgements
We apologize to those authors whose work could not be cited owing to space
limitations.
Competing interests
The authors declare no competing or financial interests.
Author contributions
All authors contributed to the writing of the manuscript.
Funding
This research received no specific grant from any funding agency in the public,
commercial or not-for-profit sectors.
References
Ameen, N. A. and Salas, P. J. I. (2000). Microvillus inclusion disease: a genetic
defect affecting apical membrane protein traffic in intestinal epithelium. Traffic 1,
76-83.
Behrendt, M., Keiser, M., Hoch, M. and Naim, H. Y. (2009). Impaired trafficking and
subcellular localization of a mutant lactase associated with congenital lactase
deficiency. Gastroenterology 136, 2295-2303.
Bigorgne, A. E., Farin, H. F., Lemoine, R., Mahlaoui, N., Lambert, N., Gil, M.,
Schulz, A., Philippet, P., Schlesser, P., Abrahamsen, T. G. et al. (2014). TTC7A
mutations disrupt intestinal epithelial apicobasal polarity. J. Clin. Invest. 124,
328-337.
Bryant, D. M., Datta, A., Rodrı́guez-Fraticelli, A. E., Perä nen, J., Martı́nBelmonte, F. and Mostov, K. E. (2010). A molecular network for de novo
generation of the apical surface and lumen. Nat. Cell Biol. 12, 1035-1045.
Buhman, K. K., Smith, S. J., Stone, S. J., Repa, J. J., Wong, J. S., Knapp, F. F., Jr,
Burri, B. J., Hamilton, R. L., Abumrad, N. A. and Farese, R. V.Jr.(2002). DGAT1
is not essential for intestinal triacylglycerol absorption or chylomicron synthesis.
J. Biol. Chem. 277, 25474-25479.
Buzza, M. S., Netzel-Arnett, S., Shea-Donohue, T., Zhao, A., Lin, C.-Y., List, K.,
Szabo, R., Fasano, A., Bugge, T. H. and Antalis, T. M. (2010). Membraneanchored serine protease matriptase regulates epithelial barrier formation and
permeability in the intestine. Proc. Natl. Acad. Sci. USA 107, 4200-4205.
Canani, R. B. and Terrin, G. (2011). Recent progress in congenital diarrheal
disorders. Curr. Gastroenterol. Rep. 13, 257-264.
Canani, R. B., Castaldo, G., Bacchetta, R., Martı́n, M. G. and Goulet, O. (2015).
Congenital diarrhoeal disorders: advances in this evolving web of inherited
enteropathies. Nat. Rev. Gastroenterol. Hepatol. 12, 293-302.
10
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
Cartó n-Garcı́a, F., Overeem, A. W., Nieto, R., Bazzocco, S., Dopeso, H., Macaya,
I., Bilic, J., Landolfi, S., Hernandez-Losa, J., Schwartz, S. et al. (2015). Myo5b
knockout mice as a model of microvillus inclusion disease. Sci. Rep. 5, 12312.
Chograni, M., Alkuraya, F. S., Ourteni, I., Maazoul, F., Lariani, I. and Chaabouni,
H. B. (2015). Autosomal recessive congenital cataract, intellectual disability
phenotype linked to STX3 in a consanguineous Tunisian family. Clin. Genet. 88,
283-287.
Crawley, S. W., Mooseker, M. S. and Tyska, M. J. (2014). Shaping the intestinal
brush border. J. Cell Biol. 207, 441-451.
Cutz, E., Rhoads, J. M., Drumm, B., Sherman, P. M., Durie, P. R. and Forstner,
G. G. (1989). Microvillus inclusion disease: an inherited defect of brush-border
assembly and differentiation. N. Engl. J. Med. 320, 646-651.
De Boer, P., Hoogenboom, J. P. and Giepmans, B. N. G. (2015). Correlated light
and electron microscopy: ultrastructure lights up! Nat. Methods 12, 503-513.
Dhekne, H. S., Hsiao, N.-H., Roelofs, P., Kumari, M., Slim, C. L., Rings, E. H. H.
M. and van Ijzendoorn, S. C. D. (2014). Myosin Vb and Rab11a regulate
phosphorylation of ezrin in enterocytes. J. Cell. Sci. 127, 1007-1017.
Fabre, A., Charroux, B., Martinez-Vinson, C., Roquelaure, B., Odul, E., Sayar,
E., Smith, H., Colomb, V., Andre, N., Hugot, J.-P. et al. (2012). SKIV2L
mutations cause syndromic diarrhea, or trichohepatoenteric syndrome.
Am. J. Hum. Genet. 90, 689-692.
Faller, N., Gautschi, I. and Schild, L. (2014). Functional analysis of a missense
mutation in the serine protease inhibitor SPINT2 associated with congenital
sodium diarrhea. PLoS ONE 9, e94267.
Fiskerstrand, T., Arshad, N., Haukanes, B. I., Tronstad, R. R., Pham, K. D.-C.,
Johansson, S., Håvik, B., Tønder, S. L., Levy, S. E., Brackman, D. et al. (2012).
Familial diarrhea syndrome caused by an activating GUCY2C mutation.
N. Engl. J. Med. 366, 1586-1595.
Forster, R., Chiba, K., Schaeffer, L., Regalado, S. G., Lai, C. S., Gao, Q., Kiani,
S., Farin, H. F., Clevers, H., Cost, G. J. et al. (2014). Human intestinal tissue with
adult stem cell properties derived from pluripotent stem cells. Stem Cell Rep. 2,
838-852.
Giepmans, B. N. G. and van Ijzendoorn, S. C. D. (2009). Epithelial cell-cell
junctions and plasma membrane domains. Biochim. Biophys. Acta 1788,
820-831.
Girard, M., Lacaille, F., Verkarre, V., Mategot, R., Feldmann, G., Grodet, A.,
Sauvat, F., Irtan, S., Davit-Spraul, A., Jacquemin, E. et al. (2014). MYO5B and
bile salt export pump contribute to cholestatic liver disorder in microvillous
inclusion disease. Hepatology 60, 301-310.
Golachowska, M. R., Hoekstra, D. and van IJzendoorn, S. C. D. (2010). Recycling
endosomes in apical plasma membrane domain formation and epithelial cell
polarity. Trends Cell Biol. 20, 618-626.
Golachowska, M. R., van Dael, C. M. L., Keuning, H., Karrenbeld, A., Hoekstra,
D., Gijsbers, C. F. M., Benninga, M. A., Rings, E. H. H. M. and van Ijzendoorn,
S. C. D. (2012). MYO5B mutations in patients with microvillus inclusion disease
presenting with transient renal Fanconi syndrome. J. Pediatr. Gastroenterol. Nutr.
54, 491-498.
Golin-Bisello, F., Bradbury, N. and Ameen, N. (2005). STa and cGMP stimulate
CFTR translocation to the surface of villus enterocytes in rat jejunum and is
regulated by protein kinase G. Am. J. Physiol. Cell Physiol. 289, C708-C716.
Goulet, O., Salomon, J., Ruemmele, F., de Serres, N. P.-M. and Brousse, N.
(2007). Intestinal epithelial dysplasia (tufting enteropathy). Orphanet J. Rare Dis.
2, 20.
Goulet, O., Vinson, C., Roquelaure, B., Brousse, N., Bodemer, C. and Cé zard,
J.-P. (2008). Syndromic ( phenotypic) diarrhea in early infancy. Orphanet J. Rare
Dis. 3, 6.
Groisman, G. M., Ben-Izhak, O., Schwersenz, A., Berant, M. and Fyfe, B. (1993).
The value of polyclonal carcinoembryonic antigen immunostaining in the
diagnosis of microvillous inclusion disease. Hum. Pathol. 24, 1232-1237.
Guerra, E., Lattanzio, R., La Sorda, R., Dini, F., Tiboni, G. M., Piantelli, M. and
Alberti, S. (2012). mTrop1/Epcam knockout mice develop congenital tufting
enteropathy through dysregulation of intestinal E-cadherin/β-catenin. PLoS ONE
7, e49302.
Haas, J. T., Winter, H. S., Lim, E., Kirby, A., Blumenstiel, B., DeFelice, M.,
Gabriel, S., Jalas, C., Branski, D., Grueter, C. A. et al. (2012). DGAT1 mutation
is linked to a congenital diarrheal disorder. J. Clin. Invest. 122, 4680-4684.
Halac, U., Lacaille, F., Joly, F., Hugot, J.-P., Talbotec, C., Colomb, V.,
Ruemmele, F. M. and Goulet, O. (2011). Microvillous inclusion disease: how to
improve the prognosis of a severe congenital enterocyte disorder. J. Pediatr.
Gastroenterol. Nutr. 52, 460-465.
Hartley, J. L., Zachos, N. C., Dawood, B., Donowitz, M., Forman, J., Pollitt, R. J.,
Morgan, N. V., Tee, L., Gissen, P., Kahr, W. H. A. et al. (2010). Mutations in
TTC37 cause trichohepatoenteric syndrome ( phenotypic diarrhea of infancy).
Gastroenterology 138, 2388-2398, 2398.e1-2.
Heinz-Erian, P., Mü ller, T., Krabichler, B., Schranz, M., Becker, C.,
Rü schendorf, F., Nü rnberg, P., Rossier, B., Vujic, M., Booth, I. W. et al.
(2009). Mutations in SPINT2 cause a syndromic form of congenital sodium
diarrhea. Am. J. Hum. Genet. 84, 188-196.
Jones, B., Jones, E. L., Bonney, S. A., Patel, H. N., Mensenkamp, A. R.,
Eichenbaum-Voline, S., Rudling, M., Myrdal, U., Annesi, G., Naik, S. et al.
Disease Models & Mechanisms
CLINICAL PUZZLE
(2003). Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid
absorption disorders. Nat. Genet. 34, 29-31.
Kammermeier, J., Drury, S., James, C. T., Dziubak, R., Ocaka, L., Elawad, M.,
Beales, P., Lench, N., Uhlig, H. H., Bacchelli, C. et al. (2014). Targeted gene
panel sequencing in children with very early onset inflammatory bowel disease–
evaluation and prospective analysis. J. Med. Genet. 51, 748-755.
Knowles, B. C., Roland, J. T., Krishnan, M., Tyska, M. J., Lapierre, L. A.,
Dickman, P. S., Goldenring, J. R. and Shub, M. D. (2014). Myosin Vb
uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease. J. Clin.
Invest. 124, 2947-2962.
Knowles, B. C., Weis, V. G., Yu, S., Roland, J. T., Williams, J. A., Alvarado, G. S.,
Lapierre, L. A., Shub, M. D., Gao, N. and Goldenring, J. R. (2015). Rab11a
regulates syntaxin 3 localization and microvillus assembly in enterocytes. J. Cell.
Sci. 128, 1617-1626.
Kozan, P. A., McGeough, M. D., Peña, C. A., Mueller, J. L., Barrett, K. E.,
Marchelletta, R. R. and Sivagnanam, M. (2015). Mutation of EpCAM leads to
intestinal barrier and ion transport dysfunction. J. Mol. Med. 93, 535-545.
Kravtsov, D., Mashukova, A., Forteza, R., Rodriguez, M. M., Ameen, N. A. and
Salas, P. J. (2014). Myosin 5b loss of function leads to defects in polarized
signaling: implication for microvillus inclusion disease pathogenesis and
treatment. Am. J. Physiol. Gastrointest. Liver Physiol. 307, G992-G1001.
Kumichel, A., Kapp, K. and Knust, E. (2015). A conserved di-basic motif of
Drosophila crumbs contributes to efficient ER export. Traffic 16, 604-616.
Kuokkanen, M., Kokkonen, J., Enattah, N. S., Ylisaukko-Oja, T., Komu, H.,
Varilo, T., Peltonen, L., Savilahti, E. and Jä rvelä , I. (2006). Mutations in the
translated region of the lactase gene (LCT) underlie congenital lactase deficiency.
Am. J. Hum. Genet. 78, 339-344.
Ledoussal, C., Woo, A. L., Miller, M. L. and Shull, G. E. (2001). Loss of the NHE2
Na(+)/H(+) exchanger has no apparent effect on diarrheal state of NHE3-deficient
mice. Am. J. Physiol. Gastrointest. Liver Physiol. 281, G1385-G1396.
Lemale, J., Coulomb, A., Dubern, B., Boudjemaa, S., Viola, S., Josset, P.,
Tounian, P. and Girardet, J.-P. (2011). Intractable diarrhea with tufting
enteropathy: a favorable outcome is possible. J. Pediatr. Gastroenterol. Nutr.
52, 734-739.
Levic, D. S., Minkel, J. R., Wang, W.-D., Rybski, W. M., Melville, D. B. and
Knapik, E. W. (2015). Animal model of Sar1b deficiency presents lipid absorption
deficits similar to Anderson disease. J. Mol. Med. 93, 165-176.
Levy, E., Harmel, E., Laville, M., Sanchez, R., Emonnot, L., Sinnett, D., Ziv, E.,
Delvin, E., Couture, P., Marcil, V. et al. (2011). Expression of Sar1b enhances
chylomicron assembly and key components of the coat protein complex II system
driving vesicle budding. Arterioscler. Thromb. Vasc. Biol. 31, 2692-2699.
Mansbach, C. M. and Siddiqi, S. A. (2010). The biogenesis of chylomicrons. Annu.
Rev. Physiol. 72, 315-333.
Marchiando, A. M., Graham, W. V. and Turner, J. R. (2010). Epithelial barriers in
homeostasis and disease. Annu. Rev. Pathol. 5, 119-144.
Martı́n, M. G., Turk, E., Lostao, M. P., Kerner, C. and Wright, E. M. (1996). Defects
in Na+/glucose cotransporter (SGLT1) trafficking and function cause glucosegalactose malabsorption. Nat. Genet. 12, 216-220.
Martin, B. A., Kerner, J. A., Hazard, F. K. and Longacre, T. A. (2014). Evaluation
of intestinal biopsies for pediatric enteropathy: a proposed immunohistochemical
panel approach. Am. J. Surg. Pathol. 38, 1387-1395.
Massarwa, R., Schejter, E. D. and Shilo, B.-Z. (2009). Apical secretion in epithelial
tubes of the Drosophila embryo is directed by the Formin-family protein
Diaphanous. Dev. Cell 16, 877-888.
Massey-Harroche, D. (2000). Epithelial cell polarity as reflected in enterocytes.
Microsc. Res. Tech. 49, 353-362.
Matano, M., Date, S., Shimokawa, M., Takano, A., Fujii, M., Ohta, Y., Watanabe,
T., Kanai, T. and Sato, T. (2015). Modeling colorectal cancer using CRISPRCas9-mediated engineering of human intestinal organoids. Nat. Med. 21,
256-262.
Melendez, J., Liu, M., Sampson, L., Akunuru, S., Han, X., Vallance, J., Witte, D.,
Shroyer, N. and Zheng, Y. (2013). Cdc42 coordinates proliferation, polarity,
migration, and differentiation of small intestinal epithelial cells in mice.
Gastroenterology 145, 808-819.
Mierau, G. W., Wills, E. J., Wyatt-Ashmead, J., Hoffenberg, E. J. and Cutz, E.
(2001). Microvillous inclusion disease: report of a case with atypical features.
Ultrastruct Pathol 25, 517-521.
Mouzaki, M., Vresk, L. and Gonska, T. (2014). An infant with vomiting, diarrhea,
and failure to thrive. Chylomicron retention disease. Gastroenterology 146, 912,
1137-1138.
Mü ller, T., Hess, M. W., Schiefermeier, N., Pfaller, K., Ebner, H. L., Heinz-Erian,
P., Ponstingl, H., Partsch, J., Rö llinghoff, B., Kö hler, H. et al. (2008). MYO5B
mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.
Nat. Genet. 40, 1163-1165.
Overeem, A. W., Bryant, D. M. and van IJzendoorn, S. C. D. (2015). Mechanisms
of apical-basal axis orientation and epithelial lumen positioning. Trends Cell Biol.
25, 476-485.
Perry, A., Bensallah, H., Martinez-Vinson, C., Berrebi, D., Arbeille, B., Salomon,
J., Goulet, O., Marinier, E., Drunat, S., Samson-Bouma, M.-E. et al. (2014).
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
Microvillous atrophy: atypical presentations. J. Pediatr. Gastroenterol. Nutr. 59,
779-785.
Phillips, A. D., Szafranski, M., Man, L.-Y. and Wall, W. J. (2000). Periodic acidSchiff staining abnormality in microvillous atrophy: photometric and ultrastructural
studies. J. Pediatr. Gastroenterol. Nutr. 30, 34-42.
Planès, C. and Caughey, G. H. (2007). Regulation of the epithelial Na+ channel by
peptidases. Curr. Top. Dev. Biol. 78, 23-46.
Rhoads, J. M., Vogler, R. C., Lacey, S. R., Reddick, R. L., Keku, E. O., Azizkhan,
R. G. and Berschneider, H. M. (1991). Microvillus inclusion disease. In vitro
jejunal electrolyte transport. Gastroenterology 100, 811-817.
Riento, K., Kauppi, M., Keranen, S. and Olkkonen, V. M. (2000). Munc18-2, a
functional partner of syntaxin 3, controls apical membrane trafficking in epithelial
cells. J. Biol. Chem. 275, 13476-13483.
Ritz, V., Alfalah, M., Zimmer, K.-P., Schmitz, J., Jacob, R. and Naim, H. Y. (2003).
Congenital sucrase-isomaltase deficiency because of an accumulation of the
mutant enzyme in the endoplasmic reticulum. Gastroenterology 125, 1678-1685.
Ruemmele, F. M., Mü ller, T., Schiefermeier, N., Ebner, H. L., Lechner, S., Pfaller,
K., Thö ni, C. E., Goulet, O., Lacaille, F., Schmitz, J. et al. (2010). Loss-offunction of MYO5B is the main cause of microvillus inclusion disease: 15 novel
mutations and a CaCo-2 RNAi cell model. Hum. Mutat. 31, 544-551.
Sakamori, R., Das, S., Yu, S., Feng, S., Stypulkowski, E., Guan, Y., Douard, V.,
Tang, W., Ferraris, R. P., Harada, A. et al. (2012). Cdc42 and Rab8a are critical
for intestinal stem cell division, survival, and differentiation in mice. J. Clin. Invest.
122, 1052-1065.
Saotome, I., Curto, M. and McClatchey, A. I. (2004). Ezrin is essential for epithelial
organization and villus morphogenesis in the developing intestine. Dev. Cell 6,
855-864.
Sato, T., Mushiake, S., Kato, Y., Sato, K., Sato, M., Takeda, N., Ozono, K., Miki,
K., Kubo, Y., Tsuji, A. et al. (2007). The Rab8 GTPase regulates apical protein
localization in intestinal cells. Nature 448, 366-369.
Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange,
D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J. et al. (2009). Single Lgr5
stem cells build crypt-villus structures in vitro without a mesenchymal niche.
Nature 459, 262-265.
Schnell, U., Kuipers, J., Mueller, J. L., Veenstra-Algra, A., Sivagnanam, M. and
Giepmans, B. N. G. (2013a). Absence of cell-surface EpCAM in congenital tufting
enteropathy. Hum. Mol. Genet. 22, 2566-2571.
Schnell, U., Cirulli, V. and Giepmans, B. N. G. (2013b). EpCAM: structure and
function in health and disease. Biochim. Biophys. Acta 1828, 1989-2001.
Shifrin, D. A., McConnell, R. E., Nambiar, R., Higginbotham, J. N., Coffey, R. J.
and Tyska, M. J. (2012). Enterocyte microvillus-derived vesicles detoxify
bacterial products and regulate epithelial-microbial interactions. Curr. Biol. 22,
627-631.
Shillingford, N. M., Calicchio, M. L., Teot, L. A., Boyd, T., Kurek, K. C.,
Goldsmith, J. D., Bousvaros, A., Perez-Atayde, A. R. and Kozakewich,
H. P. W. (2015). Villin immunohistochemistry is a reliable method for diagnosing
microvillus inclusion disease. Am. J. Surg. Pathol. 39, 245-250.
Sivagnanam, M., Mueller, J. L., Lee, H., Chen, Z., Nelson, S. F., Turner, D.,
Zlotkin, S. H., Pencharz, P. B., Ngan, B.-Y., Libiger, O. et al. (2008).
Identification of EpCAM as the gene for congenital tufting enteropathy.
Gastroenterology 135, 429-437.
Slanchev, K., Carney, T. J., Stemmler, M. P., Koschorz, B., Amsterdam, A.,
Schwarz, H. and Hammerschmidt, M. (2009). The epithelial cell adhesion
molecule EpCAM is required for epithelial morphogenesis and integrity during
zebrafish epiboly and skin development. PLoS Genet. 5, e1000563.
Sobajima, T., Yoshimura, S.-I., Iwano, T., Kunii, M., Watanabe, M., Atik, N.,
Mushiake, S., Morii, E., Koyama, Y., Miyoshi, E. et al. (2014). Rab11a is
required for apical protein localisation in the intestine. Biol. Open. 4, 86-94.
Sonal, Sidhaye, J., Phatak, M., Banerjee, S., Mulay, A., Deshpande, O., Bhide,
S., Jacob, T., Gehring, I., Nuesslein-Volhard, C. et al. (2014). Myosin Vb
mediated plasma membrane homeostasis regulates peridermal cell size and
maintains tissue homeostasis in the zebrafish epidermis. PLoS Genet. 10,
e1004614.
Spence, J. R., Mayhew, C. N., Rankin, S. A., Kuhar, M. F., Vallance, J. E., Tolle,
K., Hoskins, E. E., Kalinichenko, V. V., Wells, S. I., Zorn, A. M. et al. (2011).
Directed differentiation of human pluripotent stem cells into intestinal tissue in
vitro. Nature 470, 105-109.
Stepensky, P., Bartram, J., Barth, T. F., Lehmberg, K., Walther, P., Amann, K.,
Philips, A. D., Beringer, O., Zur Stadt, U., Schulz, A. et al. (2013). Persistent
defective membrane trafficking in epithelial cells of patients with familial
hemophagocytic lymphohistiocytosis type 5 due to STXBP2/MUNC18-2
mutations. Pediatr. Blood Cancer 60, 1215-1222.
Szabo, R., Hobson, J. P., Christoph, K., Kosa, P., List, K. and Bugge, T. H.
(2009). Regulation of cell surface protease matriptase by HAI2 is essential for
placental development, neural tube closure and embryonic survival in mice.
Development 136, 2653-2663.
Szperl, A. M., Golachowska, M. R., Bruinenberg, M., Prekeris, R., Thunnissen,
A.-M. W. H., Karrenbeld, A., Dijkstra, G., Hoekstra, D., Mercer, D., Ksiazyk, J.
et al. (2011). Functional characterization of mutations in the myosin Vb gene
11
Disease Models & Mechanisms
CLINICAL PUZZLE
CLINICAL PUZZLE
Wedenoja, S., Pekansaari, E., Hö glund, P., Mä kelä , S., Holmberg, C. and Kere,
J. (2011). Update on SLC26A3 mutations in congenital chloride diarrhea. Hum.
Mutat. 32, 715-722.
Weisz, O. A. and Rodriguez-Boulan, E. (2009). Apical trafficking in epithelial cells:
signals, clusters and motors. J. Cell. Sci. 122, 4253-4266.
Whiteman, E. L., Fan, S., Harder, J. L., Walton, K. D., Liu, C.-J., Soofi, A., Fogg,
V. C., Hershenson, M. B., Dressler, G. R., Deutsch, G. H. et al. (2014). Crumbs3
is essential for proper epithelial development and viability. Mol. Cell. Biol. 34,
43-56.
Wiegerinck, C. L., Janecke, A. R., Schneeberger, K., Vogel, G. F., van HaaftenVisser, D. Y., Escher, J. C., Adam, R., Thö ni, C. E., Pfaller, K., Jordan, A. J.
et al. (2014). Loss of syntaxin 3 causes variant microvillus inclusion disease.
Gastroenterology 147, 65-68.e10.
Winter, J. F., Hö pfner, S., Korn, K., Farnung, B. O., Bradshaw, C. R., Marsico, G.,
Volkmer, M., Habermann, B. and Zerial, M. (2012). Caenorhabditis elegans
screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and
apicobasal cell polarity. Nat. Cell Biol. 14, 666-676.
Yang, Y., Tomkovich, S. and Jobin, C. (2014). Could a swimming creature inform
us on intestinal diseases? Lessons from zebrafish. Inflamm. Bowel Dis. 20,
956-966.
Young, S. G., Hubl, S. T., Smith, R. S., Snyder, S. M. and Terdiman, J. F. (1990).
Familial hypobetalipoproteinemia caused by a mutation in the apolipoprotein B
gene that results in a truncated species of apolipoprotein B (B-31). A unique
mutation that helps to define the portion of the apolipoprotein B molecule required
for the formation of buoyant, triglyceride-rich lipoproteins. J. Clin. Invest. 85,
933-942.
Yui, S., Nakamura, T., Sato, T., Nemoto, Y., Mizutani, T., Zheng, X., Ichinose, S.,
Nagaishi, T., Okamoto, R., Tsuchiya, K. et al. (2012). Functional engraftment of
+
colon epithelium expanded in vitro from a single adult Lgr5 stem cell. Nat. Med.
18, 618-623.
zur Stadt, U., Rohr, J., Seifert, W., Koch, F., Grieve, S., Pagel, J., Strauss, J.,
Kasper, B., Nü rnberg, G., Becker, C. et al. (2009). Familial hemophagocytic
lymphohistiocytosis type 5 (FHL-5) is caused by mutations in Munc18-2 and
impaired binding to syntaxin 11. Am. J. Hum. Genet. 85, 482-492.
Disease Models & Mechanisms
associated with microvillus inclusion disease. J. Pediatr. Gastroenterol. Nutr. 52,
307-313.
Thoeni, C. E., Vogel, G. F., Tancevski, I., Geley, S., Lechner, S., Pfaller, K., Hess,
M. W., Mü ller, T., Janecke, A. R., Avitzur, Y. et al. (2014). Microvillus inclusion
disease: loss of Myosin vb disrupts intracellular traffic and cell polarity. Traffic 15,
22-42.
Van der Velde, K. J., Dhekne, H. S., Swertz, M. A., Sirigu, S., Ropars, V., Vinke,
P. C., Rengaw, T., van den Akker, P. C., Rings, E. H. H. M., Houdusse, A. et al.
(2013). An overview and online registry of microvillus inclusion disease patients
and their MYO5B mutations. Hum. Mutat. 34, 1597-1605.
Van Der Werf, C. S., Wabbersen, T. D., Hsiao, N.-H., Paredes, J., Etchevers,
H. C., Kroisel, P. M., Tibboel, D., Babarit, C., Schreiber, R. A., Hoffenberg,
E. J. et al. (2012). CLMP is required for intestinal development, and loss-offunction mutations cause congenital short-bowel syndrome. Gastroenterology
142, 453-462.e3.
Van der Wouden, J. M., Maier, O., van IJzendoorn, S. C. D. and Hoekstra, D.
(2003). Membrane dynamics and the regulation of epithelial cell polarity. Int. Rev.
Cytol. 226, 127-164.
Van Ham, T. J., Brady, C. A., Kalicharan, R. D., Oosterhof, N., Kuipers, J.,
Veenstra-Algra, A., Sjollema, K. A., Peterson, R. T., Kampinga, H. H. and
Giepmans, B. N. G. (2014). Intravital correlated microscopy reveals differential
macrophage and microglial dynamics during resolution of neuroinflammation. Dis.
Model. Mech. 7, 857-869.
Wakabayashi, Y., Dutt, P., Lippincott-Schwartz, J. and Arias, I. M. (2005).
Rab11a and myosin Vb are required for bile canalicular formation in WIF-B9 cells.
Proc. Natl. Acad. Sci. USA 102, 15087-15092.
Wang, X., Matteson, J., An, Y., Moyer, B., Yoo, J.-S., Bannykh, S., Wilson, I. A.,
Riordan, J. R. and Balch, W. E. (2004). COPII-dependent export of cystic fibrosis
transmembrane conductance regulator from the ER uses a di-acidic exit code.
J. Cell Biol. 167, 65-74.
Watson, C. L., Mahe, M. M., Mú nera, J., Howell, J. C., Sundaram, N., Poling,
H. M., Schweitzer, J. I., Vallance, J. E., Mayhew, C. N., Sun, Y. et al. (2014). An
in vivo model of human small intestine using pluripotent stem cells. Nat. Med. 20,
1310-1314.
Disease Models & Mechanisms (2016) 9, 1-12 doi:10.1242/dmm.022269
12